Yale School of Public Health, Department of Epidemiology of Microbial Diseases, LEPH, New Haven, Connecticut, United States of America.

Abstract

The agents of sleeping sickness disease, Trypanosoma brucei complex parasites, are transmitted to mammalian hosts through the bite of an infected tsetse. Information on tsetse-trypanosome interactions in the salivary gland (SG) tissue, and on mammalian infective metacyclic (MC) parasites present in the SG, is sparse. We performed RNA-seq analyses from uninfected and T. b. brucei infected SGs of Glossina morsitans morsitans. Comparison of the SG transcriptomes to a whole body fly transcriptome revealed that only 2.7% of the contigs are differentially expressed during SG infection, and that only 263 contigs (0.6%) are preferentially expressed in the SGs (SG-enriched). The expression of only 37 contigs (0.08%) and 27 SG-enriched contigs (10%) were suppressed in infected SG. These suppressed contigs accounted for over 55% of the SG transcriptome, and included the most abundant putative secreted proteins with anti-hemostatic functions present in saliva. In contrast, expression of putative host proteins associated with immunity, stress, cell division and tissue remodeling were enriched in infected SG suggesting that parasite infections induce host immune and stress response(s) that likely results in tissue renewal. We also performed RNA-seq analysis from mouse blood infected with the same parasite strain, and compared the transcriptome of bloodstream form (BSF) cells with that of parasites obtained from the infected SG. Over 30% of parasite transcripts are differentially regulated between the two stages, and reflect parasite adaptations to varying host nutritional and immune ecology. These differences are associated with the switch from an amino acid based metabolism in the SG to one based on glucose utilization in the blood, and with surface coat modifications that enable parasite survival in the different hosts. This study provides a foundation on the molecular aspects of the trypanosome dialogue with its tsetse and mammalian hosts, necessary for future functional investigations.

A. Number of RNA-seq reads after quality control and removal of parasite reads. B. Reads per contigs for control salivary gland. C. Contigs enriched in salivary glands (SG-enriched data set) in relation to whole female fly transcriptome data. D. Contigs with higher or lower expression following parasite infection in the salivary glands. E. The abundance of reads and fold difference of expression per contig are shown from parasite infected (shown in red) and from normal (shown in white) salivary glands. Those denoted below the line indicate reduced expression while those above indicate increased expression. The red dots below the line indicate the 21 secreted peptide contigs that correspond to 56.8% of total contigs.

A. Fold change during salivary gland infection based on RNA-seq analysis from SG-enriched dataset that have a combined infected and control RPKM value of over 1000 from . The combined infected and control RPKM value is shown next to each bar. B. Percent of RNA-seq reads represented by the contigs with decreased expression during trypanosome infection. C. Total protein profile analyzed by SDS-PAGE analysis from one pair of infected and control salivary glands, one representative sample is shown. D. Western blot analysis of Tsal1, TSGF-1, TSGF-2 as well as Tubulin from control and infected SG.